The long term goal of this proposal is to understand the role of specific caspase cleavage events in driving apoptosis. Apoptosis is an altruistic process for removing infected, DNA damaged, or precancerous cells. The final steps are driven by a class of intracellular cysteine proteases, known as caspases, that deconstruct the cell by specific (and usually single) cleavage of up to 1000 proteins in human cells. The targets of proteolysis teach us much about cellular pathways and networks that maintain homeostasis as well as the apoptotic machinery that drives the process. Our hypothesis is that many targets of apoptosis form functional webs or struts that when cleaved alone can trigger apoptosis. Unfortunately, given that so many targets are cleaved simultaneously by caspases, the importance of individual proteolytic events can not be assessed. To systematically attack this problem we have developed a platform of technologies that allows us to begin to dissect the importance of cutting individual targets in driving apoptosis. These include the development of a site-specific protease (SNIPer) which is activated by a small molecule (rapamycin) and cleaves single targets containing its specific recognition sequence that is not found in the human proteome. The second is a post- translational gene replacement vector, which enables us to introduce the target gene of interest with a SNIPer site replacing a caspase site and simultaneously expressing an shRNA into the endogenous caspase target. This allows rapid and effective replacement of the endogenous caspase sensitive allele, with a specific SNIPer sensitive allele. A third technology permits us to follow the detailed events of proteolysis using a proteomic method established in our lab for tagging newly created N-termini during proteolysis. We will apply these technologies on three mini-networks that are triggered by caspase proteolysis and are thought to be critical drivers and hallmarks of apoptosis including: activation of DNA damage and inhibition of DNA repair, signaling enzymes that concentrate in the nucleus following caspase proteolysis, and subunits in the 26S proteasome that are cleaved during apoptosis and disable the proteasome which clears activated caspases. Specific Aim #1: Determine the biochemical and cellular consequences for site-specific proteolytic activation of the caspase activated DNase (CAD) and neighboring repair enzymes in apoptosis. Specific Aim#2: Determine the biochemical and cellular consequences of site-specific proteolysis of Abl kinase and CDC25A phosphatase. Specific Aim#3: Determine the biochemical and cellular consequences of caspase-like cleavages of the 19S regulatory particle of the 26S proteasome. These experiments should greatly enhance our understanding of how specific proteolytic events can spark, sensitize, and drive apoptosis. The ignition of signaling events via small molecule regulated, site-selective proteolysis sets a new paradigm for dissecting complex protease signaling pathways.